Protein activity has been photomodulated through a variety of means in living cells, including site-specific derivatization with photocleavable moieties (Goeldner and Givens, Dynamic Studies in Biology: Phototriggers, Photoswitches and Caged Biomolecules. (Wiley-VCH, 2005)) or photomodulation of small molecule ligands (Fortin et al., Nature Methods 5:331 (2008)). These techniques suffer from some combination of disadvantages preventing widespread application to intracellular proteins—irreversible activation, irradiation with toxic UV light, and/or the need to introduce caged proteins through mechanical disruption of the cell membrane.
Recent NMR studies by Harper et al. revealed the mechanism of a protein light switch in Avena sativa (oat) Phototropin1 (Harper et al., Science 301:1541 (2003); Yao et al., Nature Chemical Biology 4:491 (2008)). The switch consists of a flavin-binding LOV2 (light, oxygen or voltage) domain that interacts with a C-terminal helical extension (Jα) in the dark. Photon absorption results in formation of a covalent bond between Cys450 and the flavin chromophore, causing conformational changes that propagate through the LOV domain, resulting in dissociation and unwinding of the Jα helix.
The present invention addresses previous shortcomings in the art by providing a strategy that enables genetic encoding of the caged protein for ready introduction into cells with reversible activation at less toxic wavelengths.